Proteins acquire their unique functions through specific folding of their linear polypeptide chains. Misfolding results in numerous diseases, such as cystic fibrosis and various neurodegenerative disorders. Although a great deal of work has been done on the investigation of how the secondary and tertiary structures of proteins are formed, and both experimental and theoretical techniques for studying protein folding are continually becoming more refined, a quantitative and predictive understanding of protein folding is still not attainable. There are still many fundamental questions as to how specific and nonspecific interactions determine the protein folding pathways, the native and nonnative structures, and thermally and kinetically accessible conformation substates, and on what range of timescales do particular folding events occur. Addressing these questions presents the need for further studies with time-resolved spectroscopic techniques that can provide the necessary time resolution and structure sensitivities. The principal objective of the proposed research is therefore to develop new spectroscopic methods and new conformation probes that can be used to generate detailed structure interpretations of the transient folding species and their dynamics over the time range of interest. A detailed set of experiments are planned to gain detailed insight into the understanding of various aspects of the folding problem, including the helix-coil transition, early folding events and intermediates, and the mechanism of beta-sheet formation. The technical goals are: (a) to develop a nanosecond temperature jump infrared spectrometer that can measure both transient kinetics at discrete frequencies and time-resolved spectra at discrete reaction times; (b) to develop a microsecond FTIR coupled continuous-flow mixing apparatus; (c) to explore novel isotope editing techniques to permit site specific conformation studies; (d) to introduce nitriles into various amino acids as infrared probes of protein folding, dynamics and interactions; (e) to study the helix-coil transition; (f) to study the beta-sheet formation in the WW domain and its interaction with peptides; (g) to study the folding and spontaneous fluctuation of single GFP molecules.